U.S. patent number 7,737,089 [Application Number 10/540,396] was granted by the patent office on 2010-06-15 for photoactivatable two-stage protective groups for the synthesis of biopolymers.
This patent grant is currently assigned to Febit Holding GmbH. Invention is credited to Barbro Beijer, Ramon Guimil, Matthias Scheffler.
United States Patent |
7,737,089 |
Guimil , et al. |
June 15, 2010 |
Photoactivatable two-stage protective groups for the synthesis of
biopolymers
Abstract
The present invention relates to a process for synthesizing
biopolymers by stepwise assembly from protected synthesis building
blocks which carry two-stage protective groups. The two-stage
protective groups are activated by a first illumination step and
eliminated by a subsequent chemical treatment step.
Photoactivatable components which considerably speed up the
activation process via intramolecular triplet sensitizers or/and
have fluorescence properties are used. The two-stage protective
groups can be used in particular within the framework of quality
control.
Inventors: |
Guimil; Ramon (Heidelberg,
DE), Scheffler; Matthias (Hirschberg/Leutershausen,
DE), Beijer; Barbro (Nussloch, DE) |
Assignee: |
Febit Holding GmbH (Heidelberg,
DE)
|
Family
ID: |
32683475 |
Appl.
No.: |
10/540,396 |
Filed: |
December 23, 2003 |
PCT
Filed: |
December 23, 2003 |
PCT No.: |
PCT/EP03/14822 |
371(c)(1),(2),(4) Date: |
September 21, 2005 |
PCT
Pub. No.: |
WO2004/058391 |
PCT
Pub. Date: |
July 15, 2004 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060111564 A1 |
May 25, 2006 |
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Foreign Application Priority Data
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Dec 23, 2002 [DE] |
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102 60 591 |
Dec 23, 2002 [DE] |
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102 60 592 |
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Current U.S.
Class: |
506/41;
536/25.32; 536/25.3; 435/68.1 |
Current CPC
Class: |
B82Y
30/00 (20130101); C40B 50/18 (20130101); B01J
19/0046 (20130101); B01J 2219/00722 (20130101); B01J
2219/00596 (20130101); B01J 2219/00576 (20130101); B01J
2219/00711 (20130101); B01J 2219/00659 (20130101) |
Current International
Class: |
C40B
70/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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101 22 357 |
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Nov 2002 |
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DE |
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WO 00/66259 |
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Nov 2000 |
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WO |
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WO 02/20150 |
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Mar 2002 |
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WO |
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WO 02/62815 |
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Aug 2002 |
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WO |
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WO 03/04510 |
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Jan 2003 |
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WO |
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WO 03/004510 |
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Jan 2003 |
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WO |
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WO 2004/058392 |
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Jul 2004 |
|
WO |
|
Other References
Happ et al., "New Trityl-Based Protecting Groups with a Mild
Two-Step Removal", Nucleosides & Nucleotides, vol. 7, No. 5/6,
1988 pp. 813-816. cited by other.
|
Primary Examiner: Low; Christopher
Assistant Examiner: Gross; Christopher M
Attorney, Agent or Firm: Rothwell, Figg, Ernst &
Manbeck, P.C.
Claims
The invention claimed is:
1. A process for synthesizing biopolymers by stepwise assembly from
synthesis building blocks which carry protective groups, where at
least one synthesis building block which carries a two-stage
protective group is used, where the two-stage protective group is a
derivatized trityl group which is activated by an illumination step
and eliminated by a subsequent chemical treatment step, wherein the
activation takes place by elimination of a photoactivatable
protective group which is selected from the group consisting of
triplet-sensitized photoactivatable groups, labeled
photoactivatable groups and both.
2. The process as claimed in claim 1, wherein the chemical
treatment step comprises a treatment selected from the group
consisting of a treatment with base, a treatment with acid, an
oxidation, a reduction, a catalyzed-reaction and any combination
thereof.
3. The process as claimed in claim 2, characterized in that the
chemical treatment step comprises an acid treatment.
4. The process as claimed in claim 1, characterized in that the
synthesis building block with the two-stage protective group has
the general formula (I): ##STR00008## where R.sub.1 and R.sub.2 are
each independently selected from hydrogen, (L)-R.sub.3,
--O-(L)-R.sub.3, N(R.sub.3).sub.2, NHZ and M, R.sub.3 is a
C.sub.1-C.sub.8 alkyl group, a C.sub.2-C.sub.8-alkenyl group, a
C.sub.2-C.sub.8-alkynyl group, a C.sub.6-C.sub.25-aryl group or/and
a C.sub.5-C.sub.25-heteroaryl group, which may optionally have
substituents, L is a linker group which is optionally present, X is
the synthesis building block, M is in each case independently a
label optionally linked via a linker group, and m is in each case
independently an integer from 0 to 4, Y is in each case
independently a photoactivatable protective group as claimed in
claim 1, Z is an amino protective group, and where R.sub.1 or/and
R.sub.2 may optionally be replaced by Y.
5. The process as claimed in claim 1, characterized in that the
two-stage protective group carries a plurality of labeling groups
which can be detected independently of one another.
6. The process as claimed in claim 5, characterized in that a first
label is linked to the photolabile component and a second label is
linked to the component which can be eliminated chemically.
7. The process as claimed in claim 4, characterized in that the
two-stage protective group comprises at least one fluorescent
label.
8. The process as claimed in claim 7, characterized in that a
fluorescent label is introduced on the trityl framework of a
compound (I).
9. The process as claimed in claim 1, characterized in that the
biopolymers are selected from nucleic acids, nucleic acid analogs,
peptides and saccharides.
10. The process as claimed in claim 9, characterized in that the
biopolymers are selected from nucleic acids and nucleic acid
analogs.
11. The process as claimed in claim 10, characterized in that
phosphoramidites are used as synthesis building blocks.
12. The process as claimed in claim 11, characterized in that
phosphoramidite building blocks carrying the two-stage protective
group on the 5'-O atom are used.
13. The process as claimed in claim 1, characterized in that the
synthesis of the biopolymers includes the use of spacer and/or
linker building blocks.
14. The process as claimed in claim 1, characterized in that the
synthesis of the biopolymers is carried out on a solid phase.
15. The process as claimed in claim 14, characterized in that a
location-dependent synthesis of a plurality of biopolymers is
carried out with in each case a different sequence of synthesis
building blocks on a single support.
16. The process as claimed in claim 1, characterized in that a
synthesis building block with two-stage protective group is used
for quality control.
17. The process as claimed in claim 2, wherein said catalyzed
reaction is an enzymatic reaction.
Description
CROSS REFERENCE TO RELATED APPLICATION
This application is a 35 USC .sctn.371 National Phase Entry
Application from PCT/EP2003/014822, filed Dec. 23, 2003, and
designating the United States.
The present invention relates to a process for synthesizing
biopolymers by stepwise assembly from protected synthesis building
blocks which carry two-stage protective groups. The two-stage
protective groups are activated by a first illumination step and
eliminated by a subsequent chemical treatment step.
Photoactivatable components which considerably speed up the
activation process via intramolecular triplet sensitizers or/and
have fluorescence properties are used. The two-stage protective
groups can be used in particular within the framework of quality
control.
The technology of light-controlled synthesis of biopolymers using
photolabile protective groups opens up the possibility of producing
biochips in situ by synthesis from monomeric and oligomeric
building blocks. Biochips have gained a very considerable
importance for research and diagnosis since they permit rapid and
highly parallel processing of complex biological problems. However,
chips of the highest quality are required for this, so that there
is a great interest in novel and more efficient synthetic
methods.
Photolabile nucleoside derivatives are used in the light-controlled
synthesis of nucleic acid chips. In this connection, the assembly
of the chain of nucleic acid fragments normally takes place using
phosphor-amidite synthons. The building blocks each carry a
temporary photoprotective group which can be removed by incident
light. The principle of the synthesis provides for a cyclic
sequence of condensation and deprotection steps (by light). The
efficiency with which such a light-controlled synthesis can take
place is determined essentially by the photolabile protective
groups used, in particular by the efficiency with which they can be
removed in the irradiation step. The photoprotective groups used to
date for light-controlled synthesis are normally the protective
groups NVOC (S. P. A. Fodor et al., Science 251 (1991), 767 ff.),
MeNPOC (A. C. Pease et al., Proc. Natl. Acad. Sci. 91 (1994), 5022
ff.), DMBOC (M. C. Pirrung, J. Chem. 60 (1995), 1116 ff.) and NPPOC
(A. Hassan et al., Tetrahedron 53 (1997), 4247 ff.). Further known
photolabile protective groups in nucleoside and nucleotide
chemistry are o-nitrobenzyl groups and their derivatives (cf., for
example, Pillai, Org. Photochem. 9 (1987), 225; Walker et al., J.
Am. Chem. Soc. 110 (1988), 7170). A further photolabile protective
group which has been proposed is the 2-(o-nitrophenyl)ethyl group
(Pfleiderer et al., in: "Biosphosphates and their
Analogues--Synthesis, Structure, Metabolism and Activity", ELSEVIER
Science Publishers B. V. Amsterdam (1987), 133 ff.) and derivatives
thereof (WO 97/44345 and WO 96/18634).
The photolabile protective groups currently used for
light-controlled synthesis of nucleic acids (e.g. NVOC, MeNPOC,
NPPOC) are generally distinguished by a comparatively low
absorption coefficient at the wavelength of the incident light.
Irradiation of photolabile nucleoside derivatives normally takes
place with high pressure Hg lamps at a wavelength of 365 nm. The
result of the low absorption coefficient of the photolabile
protective group used at this wavelength is that only a very small
proportion of the incident light can be utilized for excitation of
the molecules. In addition, the photolabile protective groups used
are mostly colorless derivatives. The result of this in turn is
that it is not possible during the synthesis to detect by simple
spectroscopic methods whether the photolabile protective group is
still present on the nucleoside derivative or has already been
partly or completely abstracted by the input of light. The
abstraction process can thus be followed only with difficulty or
not at all.
DE 101 32 925.6 and PCT/EP02/07389 propose the use of two-stage
protective groups, where the two-stage protective groups are
activated by an illumination step and eliminated by a subsequent
chemical treatment step. The two-stage protective groups are
preferably trityl derivatives which are coupled to a
photoactivatable protective group. The trityl derivatives may also
comprise fluorescent groups in addition.
In order to eliminate the disadvantages of the previously published
prior art, according to the present invention specific
photoactivatable groups are linked to a second component whose
elimination conditions are orthogonal to those of the
photoactivatable groups, and whose removal leads to exposure of the
actual protective group which can be eliminated by chemical means,
e.g. acid catalysis. The protective group eliminated by chemical
means, which is preferably colored or/and fluorescent, can be
employed for quality control during the synthesis of
biopolymers.
The present invention provides a novel protective group with which
the activation step is induced by light and the actual deprotection
step at the reaction site takes place by chemical means, e.g. acid
catalysis (FIG. 1). This novel protective group, and molecules
carrying this protective group, can be employed for the synthesis
of biopolymers.
One aspect of the invention is thus a process for the synthesis of
biopolymers by stepwise assembly from synthesis building blocks
which carry protective groups, with use of at least one synthesis
building block which carries a two-stage protective group which is
activated by an illumination step and is eliminated by a subsequent
chemical treatment step, using as photoactivatable group a
triplet-sensitized photoactivatable group, a labeled, e.g.
fluorescent photoactivatable group or/and a labeled, e.g.
fluorescent and triplet-sensitized photoactivatable group. The
illumination step preferably comprises the elimination of a first
photolabile component of the protective group, leaving behind a
second component of the protective group which is essentially
stable to the conditions prevailing on elimination of the first
component and which can subsequently be eliminated by a chemical
treatment step. The chemical treatment step preferably comprises a
treatment with base, a treatment with acid, an oxidation, a
reduction or/and catalytic, e.g. an enzymatic, reaction. The
chemical treatment step particularly preferably comprises an acid
treatment.
In a particularly preferred embodiment of the invention, a
derivatized trityl group is used as two-stage protective group.
Trityl groups are notable for their excellent ease of elimination,
in particular by treatment with acid. The two-stage trityl
protective groups of the invention are, by contrast, not
acid-labile but are converted into an acid-labile form only after
activation and elimination of one or more photolabile
components.
Triplet-sensitized photoactivatable groups and labeled, e.g.
fluorescent, and triplet-sensitized photoactivatable groups on the
one hand have a high molar extinction coefficient at the incident
wavelength in order to contribute to a significant increase in the
population in the triplet state, and on the other hand are able to
stabilize a tertiary free radical in the aci-nitro form via I or M
effects. This leads to an increase in the overall quantum yield of
the activation step. Labeled photoactivatable groups (without
triplet sensitization) show merely a high molar extinction
coefficient at the incident wavelength, but have no direct effect
on the activation process. All said types of photoactivatable
groups are particularly suitable in quality control, for example in
fluidic microprocessors as described for example in WO
00/13018.
Particular preference is therefore given to a synthesis building
block which has a two-stage protective group and which has the
general formula (I):
##STR00001## where R.sub.1 and R.sub.2 are each independently
selected from hydrogen, (L)-R.sub.3, O-(L)-R.sub.3,
N(R.sub.3).sub.2, NHZ and M, R.sub.3 is a C.sub.1-C.sub.8 alkyl
group, a C.sub.2-C.sub.8-alkenyl group, a C.sub.2-C.sub.8-alkynyl
group, a C.sub.6-C.sub.25 aryl group or/and a
C.sub.5-C.sub.25-heteroaryl group, each of which may optionally
have one or more substituents, L is a linker group which is
optionally present, which is for example --(CH.sub.2).sub.n--,
--(CH.sub.2).sub.n--COO--, --(CH.sub.2).sub.n--CONH-- or
--(CH.sub.2).sub.n--SO.sub.2O--, --(CH.sub.2).sub.n--O--,
--(CH.sub.2).sub.n--S-- or --(CH.sub.2).sub.n--NH--, and n is an
integer from 0 to 20, M is in each case independently a label
optionally linked via a linker group L (as defined above), and m is
in each case independently an integer from 0 to 4, preferably 0, 1
or 2, X is the synthesis building block, Y is in each case
independently a photoactivatable protective group as indicated
above, Z is an amino protective group, and where R.sub.1 or/and
R.sub.2 may optionally be replaced by Y.
The alkyl, alkenyl and alkynyl groups may be linear or cyclic,
straight-chain or branched. The aryl or heteroaryl groups, e.g. N-,
O- or/and S-heteroaryl groups, may be mono- or polycyclic. Examples
of substituents of alkyl, alkenyl, alkynyl, aryl and heteroaryl
groups are halogen, e.g. F, Cl, Br, I, OH, SH, --O--, --S--,
--S(O).sub.2--, NO.sub.2, CN, COOH, CO--C.sub.1-C.sub.8-alkyl,
COO--C.sub.1-C.sub.8-alkyl, OCO--C.sub.1-C.sub.8-alkyl,
CONH--C.sub.1-C.sub.8-alkyl, CON--(C.sub.1-C.sub.8)-alkyl) 2,
C.sub.1-C.sub.8-alkyl, C.sub.2-C.sub.8-alkenyl,
C.sub.2-C.sub.8-alkynyl, C.sub.1-C.sub.8-alkoxy,
--S--C.sub.1-C.sub.8-alkyl, di(C.sub.1-C.sub.8-alkyl)amino and NHZ,
where the alkyl, alkenyl, alkynyl and alkoxycarbonyl groups may in
turn be substituted by halogen. Preferred meanings for R.sub.1 and
R.sub.2 are hydrogen, dialkylamine, e.g. N,N-dimethyl, O-methyl,
OCOO-methyl or a protective amino group, e.g. an amino group
converted into an amide function with a suitable carboxylic acid.
The number of carbon atoms in the radicals R.sub.1 and R.sub.2 of
the compound is preferably restricted to 25 in each case.
The invention also encompasses compounds which carry a plurality of
photoactivatable groups, in particular compounds of the formula (I)
in which at least one of R.sub.1 or R.sub.2 is replaced by a
photoactivatable protective group. It is preferred for 1 to 3
photoactivatable protective groups to be present. It is
additionally possible for one or more labeling groups to be present
and to be linked to the photoactivatable component or/and to the
chemically active component. Thus, one or more labeling groups in
the compounds (I) may be present at the o or/and m positions of the
phenyl rings in the trityl system.
It is possible by varying the radicals R.sub.1 and R.sub.2 and
substituting one or both radicals by photoactivatable protective
groups to adapt the acid lability to the desired requirements.
In a preferred embodiment, labeled photoactivatable groups of the
formula (II) are used:
##STR00002## in which Ar is a fused polycyclic, preferably tetra-,
penta- or hexacyclic, fluorescent aryl or heteroaryl, S.sub.1 and
S.sub.2 are each independently selected from hydrogen, a
C.sub.1-C.sub.8-alkyl group, a C.sub.2-C.sub.8-alkenyl group, a
C.sub.2-C.sub.8-alkynyl group, a C.sub.6-C.sub.25-aryl or/and a
C.sub.5-C.sub.25-heteroaryl group, each of which may optionally
have substituents, and Q is a group for linking the photolabile
component to the component which can be eliminated chemically. The
number of carbon atoms in the radicals Ar, S.sub.1 and S.sub.2 of
the compound (II) is preferably restricted to 25 in each case.
Examples of suitable fluorescent aryl radicals are
benzo[b]fluoranthrene, fluoranthrene, 9,10-diphenyl-anthracene,
acenaphthylene or pyrene.
Substituents of the groups Ar, S.sub.1 and S.sub.2 are as defined
above for the compounds of the formula (I). Q is preferably
SO.sub.2, OCO, OCS or CS.sub.2.
Particular preference is given to compounds (II) in which S.sub.1
and S.sub.2 are H, e.g. the compound PyMOC (as indicated in U.S.
Pat. No. 6,147,205).
In a further preferred embodiment there is use of a
photoactivatable group of the general formula (III)
##STR00003## in which T.sub.1, T.sub.2, T.sub.3, T.sub.4, T.sub.5
and T.sub.6 are each independently selected from hydrogen,
C.sub.1-C.sub.8-alkyl, C.sub.2-C.sub.8-alkenyl,
C.sub.2-C.sub.8-alkynyl, C.sub.1-C.sub.8-alkoxy,
C.sub.2-C.sub.8-alkoxycarbonyl, C.sub.6-C.sub.25-aryl or aryloxy
or/and C.sub.5-C.sub.25-heteroaryl or heteroaryloxy, each of which
may optionally have substituents, and T.sub.1 or/and T.sub.2 may
additionally be trialkylsilyl, and one of T.sub.3 and T.sub.4 may
be NO.sub.2, with the proviso that the other is then H, Q.sub.1 is
hydrogen, optionally substituted C.sub.1-C.sub.4-alkoxy or
di(C.sub.1-C.sub.4-alkyl)-amino, Z.sub.1 and Z.sub.2 together are
--OC(O)--, --NT.sub.7C(O)-- or --CT.sub.8=CT.sub.9, where T.sub.8
and T.sub.9 are defined as T.sub.3-T.sub.6, and T.sub.9 may
additionally be NO.sub.2, and adjacent groups, e.g. T.sub.8 and
T.sub.9, may form a 5- or 6-membered carbocyclic or heterocyclic,
saturated or unsaturated ring, and Q is a group for linking the
photolabile component to the component which can be eliminated
chemically. The number of carbon atoms in the radicals
T.sub.1-T.sub.9 of compound (III) is preferably restricted to 25 in
each case.
The possible substituents of the respective groups are in this case
as defined above for the compounds of the formula (I). Q is
preferably a group of the general formula: -Q.sub.2-C(Q.sub.3)-,
where Q.sub.2 is --O--, --S--, --CH.sub.2O-- or --CH.sub.2S--, and
Q.sub.3 is .dbd.O or .dbd.S. Examples of suitable compounds of the
formula (III) are described for example in WO 02/20150.
In yet a further preferred embodiment there is use of labeled
photoactivatable groups of the general formula (IV):
##STR00004## in which U.sub.1, U.sub.2, U.sub.4 and U.sub.5 are
each independently selected from hydrogen, halogen, NO.sub.2,
U.sub.6, (L)-U.sub.6, O-(L)-U.sub.6, N(U.sub.6).sub.2 and NHZ,
U.sub.6 is C.sub.1-C.sub.8-alkyl, C.sub.2-C.sub.8-alkenyl,
C.sub.2-C.sub.8-alkynyl, C.sub.6-C.sub.25-aryl or
C.sub.5-C.sub.25-heteroaryl, each of which may optionally have
substituents, L is a linker group which is optionally present, e.g.
as defined for the compounds (I), U.sub.3 is a labeling group
optionally linked via a linker group, e.g. as defined for the
compound (I), e.g. a fluorescent group, and Q is a group for
linking the photolabile component to the component which can be
eliminated chemically. The number of carbon atoms in the radicals
U.sub.1-U.sub.5 is preferably restricted to 25 in each case.
Adjacent radicals may optionally form a 5- or 6-membered
carbocyclic or heterocyclic, saturated or unsaturated ring.
The definition of the possible substituents on the radicals
U.sub.1, U.sub.2, U.sub.4 and U.sub.5 is as described for the
compounds (I). Q is preferably SO.sub.2, OCO, OCS, CS.sub.2,
CH.sub.2SO.sub.4, CH.sub.2OCO, CH.sub.2OCS, CH.sub.2CS.sub.2 etc.
The radical U.sub.3 preferably has the structure --O-L-NHCOM where
L is a linker having a chain length of preferably 1-10 atoms, e.g.
C atoms, and optionally heteroatoms such as O, S or N, and M is a
labeling group, e.g. a fluorescent group such as, for example, a
pyrene or coumarin group.
The compounds of this type are based on conventional O-nitrobenzyl
groups such as, for example, NPPOC, NVOC, MeNPOC, into which a
fluorophore has additionally been introduced. The resulting
photoactivatable molecule has a label but not a triplet
sensitization.
In yet a further preferred embodiment there is use of
photoactivatable groups of the formula (V) which are preferably
triplet-sensitized and optionally labeled groups:
##STR00005## in which V.sub.1, V.sub.2, V.sub.3, V.sub.4, V.sub.5
and V.sub.6 are each independently selected from hydrogen, halogen,
NO.sub.2, V.sub.7, (L)-V.sub.7, O-(L)-V.sub.7, N(V.sub.7).sub.2,
NHZ and M, where V.sub.7 is C.sub.1-C.sub.8-alkyl,
C.sub.2-C.sub.8-alkenyl, C.sub.2-C.sub.8-alkynyl,
C.sub.6-C.sub.25-aryl or C.sub.5-C.sub.25-heteroaryl, each of which
may optionally have substituents, L is a linker group which is
optionally present, e.g. as defined for the compounds (I), V.sub.5
and V.sub.6 may additionally be trialkylsilyl, M is a label
optionally linked via a linker group, and Q is a group for linking
the photolabile component to the component which can be eliminated
chemically. The number of carbon atoms in the radicals
V.sub.1-V.sub.6 is preferably restricted to 25 in each case.
Adjacent radicals may optionally form a 5- or 6-membered
carbocyclic or heterocyclic, saturated or unsaturated ring.
The radical V.sub.5 is particularly preferably an aryl, aryloxy,
heteroaryl or heteroaryloxy group which may be unsubstituted or may
have up to three substituents (as defined above). Particular
preference is given to polycyclic aryl, aryloxy, heteroaryl or
heteroaryloxy groups which show triplet sensitization and which
optionally may have an intrinsic fluorescence, especially if they
comprise four or more fused rings, e.g. pyrenes,
benzo[b]fluoranthrenes, fluoranthrenes, 9,10-diphenylanthracenes,
acenaphthylenes or corresponding oxy derivatives etc.
The invention also includes compounds which carry a plurality of
labels which are detectable independently of one another. Examples
of suitable labels are fluorescent groups, luminescent groups,
electrically detectable groups, e.g. ferrocenes, colored groups,
radioactive groups, groups detectable by NMR etc. The labels
preferably comprise at least one fluorescent group, which may be
combined with another, independently detectable fluorescent group
or another type of label as mentioned above. It is preferred for
one label to be linked to the photolabile component of the
protective group and for the other label to be linked to the
component which can be eliminated chemically, so that selective
elimination of the photolabile component can be detected by loss of
the first label but retention of the second label, and elimination
of the chemical component can be detected by loss of the second
label. For example, the invention includes compounds (I) which
carry a plurality of fluorescent groups, e.g. compounds in which Y
is a fluorescent photoprotective group or/and R.sub.3 and Z are
fluorescent groups on the trityl framework (R. Ramage, F. O. Wahl,
Tetrahedron Lett., 34 (1993), 7133) or molecules in which the
fluorescence has been introduced by substitution on the trityl
framework (J. L. Fourrey et al., Tetrahedron Lett., 28 (1987),
5157).
In a preferred embodiment, the labeling group on the component
which can be eliminated chemically is a fluorescent group, e.g. a
coumarin or pyrene group, which is coupled via a linker group, e.g.
a group as defined above, to the basic trityl framework, e.g. in p,
o or/and m position of the phenyl rings in the trityl system.
These labeling groups can be employed for quality control of
biochips. This can take place for example in biochip supports as
described in WO 00/13018. On use of fluorescent labeling groups
care must be taken that the excitation and emission wavelengths do
not interfere with the photoinduced activation.
The process of the invention is employed for the synthesis of
biopolymers, with the biopolymer to be synthesized being assembled
stepwise from a plurality of synthesis building blocks. The process
is particularly preferably employed for the synthesis of nucleic
acids, e.g. DNA or RNA. However, it should be noted that the
process is also suitable for the synthesis of other biopolymers
such as, for example, peptides, peptide-nucleic acids (PNAs) or
saccharides. The synthesis building block may be a monomeric
building block, e.g. a nucleoside derivative or a peptide
derivative, or else an oligomeric building block, e.g. a dimer or
trimer, i.e. for example a di- or trinucleoside derivative or a di-
or tripeptide derivative. The synthesis building block is
particularly preferably a phosphoramidite building block. It is,
however, also possible to use other nucleotide synthesis building
blocks, e.g. phosphate or phosphonate building blocks. A further
possibility is also to employ linker or spacer building blocks,
e.g. as phosphoramidites, as synthesis building blocks.
Particularly preferred linkers or spacers as carriers of two-stage
protective groups are described in DE 100 41 539.3.
The synthesis building blocks of the invention carrying a two-stage
protective group generally have more strongly lipophilic properties
than the synthesis building blocks used to date in the prior art.
The solubility of the synthesis building blocks, especially of the
phosphoramidite synthons, in organic solvents is increased through
this lipophilicity. The more homogeneous reaction management made
possible thereby leads to a higher coupling efficiency compared
with the pure photolabile phosphoramidite synthons. Elimination of
the colored trityl cation of the photoprotective groups of the
invention, which has a considerably higher absorption coefficient
than the elimination products in other photodeprotection processes,
also opens up the possibility of direct online process monitoring.
This leads to an improvement in the quality control of
biochips.
The trityl group of the photoprotective groups of the invention
additionally makes selective functionalization of the 5'-hydroxy
function possible. This leads to an enormous reduction in costs,
because separation of the 3'-5' isomers is dispensed with.
Particular preference is therefore given according to the present
invention to phosphoramidite building blocks which carry the
two-stage protective group on the 5'-O atom of the sugar, in
particular of the ribose or of the deoxyribose.
The synthesis of the biopolymers can be carried out in a
conventional way, for example on a solid phase. It is particularly
preferred for a plurality of biopolymers carrying a different
sequence of synthesis building blocks to be generated in situ in
the form of an array on a single support.
Yet a further aspect of the invention are compounds of the general
formula (I)
##STR00006## where R.sub.1, Y, M and m are as defined above, and X
is a synthesis building block for synthesizing biopolymers or a
leaving group, and where R.sub.1 or/and R.sub.2 may optionally be
replaced by Y.
If X is a leaving group, it is a group which can be eliminated on
reaction of the compound (I) with another compound. X is preferably
a leaving group which can be eliminated by reaction with a
nucleophile, optionally in the presence of an auxiliary base such
as pyridine. Preferred examples of X are: Cl, Br, I, tosylate,
mesylate, trifluorosulfonate etc.
##STR00007##
The diagrammatic representation of the protective group concept of
the invention is shown in FIG. 1. The synthesis building block (A)
carries a two-stage protective group (B-C). In a first illumination
step, the photolabile portion (B) of the protective group is
eliminated. The chemically labile component (C) of the protective
group is eliminated in a second chemical treatment step, e.g. by
addition of acid, so that the synthesis building block (A) is
present in active form.
FIGS. 2 and 3 show exemplary substances from a preferred class of
two-stage protective groups of the invention. They are based on the
acid-labile trityl group, but comprise in the p position of one
phenyl radical a photolabile triplet-sensitized component (V) which
reduces or completely blocks the acid sensitivity of the trityl
group. The photolabile component in FIG. 3 shows intrinsic
fluorescence. The protective group is converted into an acid-labile
form by illumination and elimination of the photolabile component
and can subsequently be eliminated in the presence of acid to
liberate the unprotected synthesis building block.
FIG. 4 shows a further exemplary substance according to the present
invention, in which, besides the photolabile protective group Y,
also a fluorescent radical (in place of R.sub.1) is coupled to the
trityl framework.
FIG. 5 shows a preferred example of a compound (II), where Q is a
group for coupling the photolabile component to the basic trityl
framework.
FIG. 6 shows a preferred example of a compound (III), where L is a
linker group and Q is a group for coupling the photolabile
component to the basic trityl framework.
* * * * *